ABSTRACTcDNAs representing the a subunit of polyomavirus enhancer binding protein 2 (PEBP2; also cafled PEA2) were isolated. The products of the cDNAs are highly homologous to that of Drosophila segmentation gene runt (run) for an N-proximal 128-amino acid region showing 66% identity. The run homology region encompasses the domain capable of binding to a specific nucleotide sequence motif and of dimerizing with the companion 13 subunit. The human AMLI gene related to t(8;21) acute myeloid leukemia also has a run homology region. Together with the 18 subunit, which increases the affinity of the a subunit to DNA without binding to DNA by itself, PEBP2 represents a newly discovered family of transcription factor. The major species of PEBP2a mRNA was expressed in T-cell lines but not in B-cell lines tested. Evidence indicated that PEBP2 functions as a transcriptional activator and is,involved in regulation of T-ceil-specific gene expression.Potential use of polyomavirus (Py) as a probe of mouse development has been recognized since it was observed that the wild-type Py did not grow in embryonal carcinoma cells but that it grew when the cells were induced to differentiate (1)(2)(3)(4)(5). Through the analysis of the Py enhancer, which determines the differentiation-dependent and cell-typespecific expression of the viral genes, a number of transcription factors involved in developmental regulation have been identified (6-10).PEBP2/PEA2, which binds to both the A and B cores of the Py enhancer (11), is undetectable in embryonal carcinoma F9 cells and becomes detectable after the cells are induced to differentiate (6,7). It specifically recognizes a consensus sequence, R/TACCRCA (R, purine), which was originally identified in the Py enhancer (11) and is also compatible with the core motif of murine leukemia virus enhancers (12,13). In addition, many T-cell-specific genes, such as T-cell receptor (TCR) a, X3, yand 8 and CD3e genes, contain potential PEBP2 binding sites, suggesting the possibility that PEBP2 is involved in T-cell-specific gene expression (13 [708][709][710][711][712][713][714]. The mutations are the same as the M2 mutation (14). For expression in mammalian cells, the coding regions of al, a2, and 831 cDNAs were cloned into the Xho I site of pCDMPy, in which the Py origin and enhancer region were deleted from pCDM8 (Invitrogen), resulting in pCDMPy-al, pCDMPy-a2, and pCDMPy-31. The bacterial expression plasmids pETal and pETa2, carrying the whole coding regions of al and a2 cDNAs, were constructed in the same way as pETf82 (16). To produce deletion mutants of a2, the whole coding region of a2 cDNA was inserted into the BamHI/HindIII fragment of plasmid pQE9 (Qiagen) resulting in pQE9-a2 encoding a fusion protein tagged with an N-terminal histidine cluster. Various deletions were introduced into pQE9-a2 using appropriate restriction sites: N94C306 encodes a region of aa 94-306; N1C226 encodes aa 1-226; N1C158 encodes aa 1-158; N80C226 encodes aa 80-226. N1C226, N1C158, and N80C226 proteins have one, two, o...

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Familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/ AML) is an autosomal dominant familial platelet disorder characterized by thrombocytopenia and a propensity to develop AML. Mutation analyses of RUNX1 in 3 families with FPD/AML showing linkage to chromosome 21q22.1 revealed 3 novel heterozygous point mutations (K83E, R135fsX177 (IVS4 ؉ 3delA), and Y260X). Functional investigations of the 7 FPD/ AML RUNX1 Runt domain point mutations described to date (2 frameshift, 2 nonsense, and 3 missense mutations) were performed. Consistent with the position of the mutations in the Runt domain at the RUNX1-DNA interface, DNA binding of all mutant RUNX1 proteins was absent or significantly decreased. In general, missense and nonsense RUNX1 proteins retained the ability to heterodimerize with PEBP2␤/CBF␤ and inhibited transactivation of a reporter gene by wild-type RUNX1. Colocalization of mutant RUNX1 and PEBP2␤/CBF␤ in the cytoplasm was observed. These results suggest that the sequestration of PEBP2␤/CBF␤ by mutant RUNX1 may cause the inhibitory effects. While haploinsufficiency of RUNX1 causes FPD/AML in some families (deletions and frameshifts), mutant RUNX1 proteins (missense and nonsense) may also inhibit wild-type RUNX1, possibly creating a higher propensity to develop leukemia. This is consistent with the hypothesis that a second mutation has to occur, either in RUNX1 or another gene, to cause leukemia among individuals harboring RUNX1 FPD/AML mutations and that the propensity to acquire these additional mutations is determined, at least partially, by the initial RUNX1 mutation. IntroductionThe most frequent mutations associated with leukemia are recurrent somatic chromosomal translocations or inversions, many of which involve the polyomavirus enhancer-binding protein or core-binding factor transcriptional regulation complex (PEBP2/CBF). Several translocations involve the ␣ subunit of this complex, the RUNX1 gene (also called AML1, CBF␣2, or PEBP2␣B) on chromosome 21q22.1 (t(8;21), t(3;21), and t(12;21)). Additionally, the ␤ subunit of the complex, PEBP2␤ also called CBF␤, is disrupted in inv(16)(p13;q22). 1 An abundance of evidence points to the existence of genes that predispose to hematologic malignancies. However, large multiple-generation families with hematologic malignancies alone are rare. 2 Only 2 loci for familial hematologic malignancies have been identified to date, 1 on chromosome 21q22.1 3 and the other on 16q22. 4,5 These loci contain RUNX1 and PEBP2␤/CBF␤, respectively.Studies of families that demonstrate single-gene inheritance for leukemia predisposition should help to identify the genes and mechanisms involved in the first steps of leukemia development. The autosomal dominant familial platelet disorder (FPD)/ AML (acute myelogenous leukemia; Online Mendelian Inheritance in Man no. 601399) is a good model to validate this hypothesis because, in addition to developing thrombocytopenia, patients show a propensity for progression to myelodysplasia and acute myeloid leuke...

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